Fertilization depends on the successful interaction of the sperm with the egg. The interactions between the gamete membranes is a fascinating process from a cell biological standpoint, beginning with the sperm contacting and attaching to the egg membrane, progressing to firm cell-cell adhesion, and culminating in membrane fusion. Our understanding of the molecular mechanisms underlying gamete membrane interactions is still very incomplete. The overall, long term goal of this project is to understand the mechanism and regulation of cell adhesion between mammalian gametes. Two sperm proteins that participate in mammalian gamete membrane interactions are members the ADAM (A Disintegrin and A Metalloprotease) family of proteins, fertilin alpha (ADAM1) and fertilin beta (ADAM2). Our determination of the key amino acids in fertilin beta that interact with the egg plasma membrane has led to the identification of potential fertilin beta binding partners on eggs. Specific Aim 1 of this proposal will build on this work with investigation of the interactions of candidate integrins with fertilin beta, including studies of eggs deficient in the beta1 integrin subunit. Our studies of fertilin a indicate that this sperm protein can use two different domains to interact with the egg membrane. Following up on this, Specific Aim 2 will determine how these domains of fertilin alpha interact with the egg membrane, including structure-function analysis and identification of binding partners. In addition to this analysis of specific molecular interactions, this project also will examine the mechanism by which sperm-egg adhesion occurs via the coordinated function of these and other gamete molecules. It has become clear that the interaction between the gametes is a complex event, likely to be mediated by multiple molecules on the sperm and egg and to occur in a step-wise fashion. We propose a model for gamete cell adhesion, based on other cell adhesion systems, in which multiple molecules participate and the interactions of gamete adhesion molecules progress to form firm adhesions between the sperm and egg. Specific Aim 3 will test aspects of this model, including studies of the roles of the egg cytoskeleton and the egg membrane protein CD9 in the strengthening of adhesions to sperm ADAMs. Results of these studies will have applications to human reproductive health, with implications for the management of infertility and fertility. This research also will also address fundamental questions in cell adhesion biology by examining this interesting, biologically relevant cell-cell interaction event.